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Contents
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WNT3A and Embryonic Development: Telling Left from Right
Powerful “organizer” signals emanating from a transient embryonic structure called the node play an important role in specifying the LR axis, thus defining a spatial, three-dimensional framework for embryonic development. Although the bone morphogenetic protein (BMP) family member, Nodal, is known to function in the node as the left determinant, little is known about the organizer signals that control its activation. Members of the WNT family of secreted signaling molecules are known to play important roles in development and, when misregulated, in diseases such as cancer. WNT3A is a particularly good candidate for an organizer molecule since it is expressed prior to LR axis formation and has powerful axis-inducing activity when overexpressed in frog and chick embryos. We hypothesized that WNT3A functions as an organizer during mouse embryogenesis. Analysis of embryos carrying targeted null alleles of Wnt3a demonstrates that it is indeed necessary for organ asymmetry. Heart looping is randomized, with approximately half of the mutants displaying normal rightward looping, and half displaying hearts that looped to the left (a condition known as situs inversus) or that remained in the midline (situs ambiguus) (Figure 1, parts A through C). Similar laterality defects were also observed in the lungs, liver, and gut, indicating that WNT3A plays an early global role in LR determination. To determine when, and where, Wnt3a is required for LR determination, we examined mutant embryos for the expression of several asymmetrically expressed genes known to regulate LR specification. In situ hybridization analyses indicated that asymmetric gene expression in the lateral plate mesoderm (a tissue that ultimately directs organ asymmetry) was absent (Figure 1, parts D and E). Furthermore, asymmetric gene expression in the mutant node was aberrant, even though general markers of the node and other axial tissues were expressed normally. In particular, Nodal was expressed in an unusually small, symmetrical, posterior domain in the mutant node. These results suggest that WNT3A specifically regulates a genetic program that controls Nodal expression in the node. Does WNT3A regulate Nodal gene expression in the node directly? WNTs activate target gene expression by stabilizing β-catenin, a multifunctional protein that possesses a transactivation domain. To determine which embryonic tissues respond directly to WNT signals, we examined the expression of a WNT/β-catenin–responsive lacZ reporter transgene in vivo. Examination of this reporter in wild-type and Wnt3a–/– mutant embryos illustrated that it is normally expressed in the node and adjacent mesoderm and that this expression is dependent on Wnt3a. These studies suggest that WNT3A signals to these tissues directly, and that it does so through β-catenin. Interestingly, recent reports indicate that Nodal expression in the node is controlled by the Notch signaling pathway (Raya A et al. Genes Dev 17: 1213–8, 2003; Krebs LT et al. Genes Dev 17: 1207–12, 2003), while the Notch ligand Delta-like1 (Dll1) is a target of the WNT/β-catenin pathway (Galceran J et al. Genes Dev 18: 2718–23, 2004; Hofmann M et al. Genes Dev 18: 2712–17, 2004). This suggests that WNT3A modulates Nodal expression indirectly, through the activation of Dll1. Indeed, we found that Dll1 expression is perturbed in the Wnt3a mutants, in a manner sufficient to explain the aberrant Nodal expression. Furthermore, we showed through genetic interaction studies that Dll1 and Wnt3a function in the same genetic pathway to regulate heart asymmetry. Since Dll1 is expressed only in the mesoderm immediately surrounding the node and not in the node itself, it appears that WNT3A directly activates Dll1 in the mesoderm, which then activates Notch signaling and Nodal expression in the adjacent node (Figure 1, part F). Figure 1. WNT signaling regulates left-right (LR) determination. (A through C) Scanning electron microscopy micrographs of ventral views of developing hearts on embryonic day 9.5 (E9.5). (A) The wild-type outflow tract (ot) loops to the right. Wnt3a–/– hearts display normal looping (B), inverted looping (situs inversus) (C), or loops that remain in the midline (situs ambiguus, not shown). Ventral-posterior views of E8.2 wild-type (D) and mutant (E) embryos processed for two-color whole-mount in situ hybridization showing Nodal (orange) and Lefty1 (purple) expression in the node (n), and overlapping Nodal (orange) and Lefty2 expression (purple) in the left lateral plate mesoderm (lpm). Note the absence of staining in the Wnt3a–/– lpm and the aberrant expression in the node. (F) WNT3A regulates LR determination and segmentation via the Delta/Notch pathway. The diagram depicts a perspective similar to that shown in (D) and (E). Wnt3a is expressed in the primitive streak and posterior node (red stippling) where it directly activates Dll1 expression in the mesoderm surrounding the node (blue arrows). In turn, DLL1 directly activates Nodal (red N) expression at the node/mesoderm boundary to set up the LR axis, and regulates segmentation (dashed arrows) in the presomitic mesoderm (psm). How LR asymmetric gene expression (green gradient) is established in the node is poorly understood, but involves node cilia. L, left; R, right; A, anterior; P, posterior; lv, left ventricle; s, somite; L1, Lefty1; L2, Lefty2. Wnt3a is also required for the formation of the repeated structures of the developing trunk known as segments or somites. These structures give rise to the skeletal muscles and vertebrae of the trunk. The number of somites, and therefore the number of vertebrae, can vary enormously among vertebrates—ranging from six in some adult frogs to several hundred in some shark species. Our demonstration that Wnt3a simultaneously controls LR axis determination and somite number through the Notch pathway reveals that Wnt3a links the body plan with fundamental morphogenetic events that are critical for determining the final shape of an organism. Given the high degree of conservation of WNT genes among metazoans (Kusserow A et al. Nature 433: 156–60, 2005), we suggest that WNTs such as WNT3A played an important role in the evolution of complex body plans.
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he formation of the body plan is a critical early step in the development of all vertebrate embryos. The anterior-posterior (AP) axis, which defines where the head and tail will form, is established first, followed by the dorsal-ventral (DV) and, lastly, the left-right (LR) axes. Superficially, vertebrates appear bilaterally symmetrical; however, this belies a profound LR asymmetry of the underlying organs. In mice, for example, the heart, stomach, and spleen are on the left side. The right lung has more lobes than the left, and the liver has a single left lobe. Mutations in humans and mice that disrupt LR positional information lead to deviations from this normal asymmetric organ arrangement and cause complex cardiovascular and gut defects that can be fatal. 